Various vectors have been previously developed for the purpose of plant transformation.
Recently, the entire genome sequences of Arabidopsis thaliana and rice (Oryza sativa) were elucidated, which moved the focus of plant genome studies from the accumulation of nucleotide sequence information to the elucidation of gene functions. For the elucidation of gene functions, experiments are absolutely necessary in which cloned DNA is transferred into a plant to analyze changes in the phenotype. If large DNA could be transferred in this operation, the study efficiency would be dramatically improved.
Thus, a number of vectors intended to transfer large DNA fragments into plants were developed. As typical examples, cosmid vectors for plant transformation were prepared, such as pOCA18 (Olszewski et al., 1988, Nucleic Acids Res. 16: 10765-10782) and pLZO3 (Lazo et al., 1991, Bio/Technology 9: 963-967). The use of a cosmid has the advantage that a lambda phage packaging reaction can be used, which allows easy cloning of relatively large genomic fragments (Sambrook J. and Russell D. W. 2001. Molecular Cloning, A Laboratory Manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA.). In cloning with a cosmid vector and a packaging reaction, the total size of the vector and the insert fragment is 40 kb-50 kb so that the size of the insert fragment is restricted within a certain range by the size of the vector and the sizes of the vector and the insert fragment inversely correlate with each other.
Vectors such as pOCA18 and pLZO3 contain elements for plant transformation such as T-DNA border sequences and a selectable marker (kanamycin resistance gene) in pRK290 (Ditta et al., 1980, Proc. Natl. Acad. Sci. USA 77: 7347-7351) which is a typical vector having an origin of replication (oriV) of an IncP plasmid that is functional in both E. coli and Agrobacterium. These vectors per se had a size of 24.3-30.1 kb, and therefore, the size of DNA that can be cloned using a packaging reaction was about 20 kb (pOCA18), or about 13-22 kb (pLZO3) on average. These vectors have an origin of replication (oriV) of an IncP plasmid, but other vectors such as pCIT103 and pCIT104 (Ma et al. 1992 Gene 117: 161-167) have an origin of replication from ColE1 in addition to an origin of replication (oriV) of an IncP plasmid. On the other hand, pC22 (Simoens et al. 1986 Nucleic Acids Res 14: 8073-8090) is a vector having an origin of replication from ColE1 and an origin of replication from an Ri plasmid. Other cosmid vectors capable of plant transformation include pMON565 (Klee et al. 1987 Mol Gen Genet 210: 282-287) and pCLD04541 (Bent et al. 1994 Science 265: 1856-1860), but they are not suitable for cloning DNA fragments of 25 kb or more because their own sizes are 24 kb and 29 kb, respectively. Other examples such as pE4 cos(16 kb, Klee et al. 1987 Mol Gen Genet 210: 282-287), pMON565, pLZ03, pOCA18, pCLD04541, pC22 and the like had a structure containing a cos site within the T-DNA.
Subsequently, the BIBAC vector (binary bacterial artificial chromosome, Hamilton U.S. Pat. No. 5,733,744, Hamilton et al., 1996, Proc. Natl. Acad. Sci. USA 93:9975-9979, Hamilton, 1997, Gene 200:107-116) was developed, which is capable of cloning DNA fragments of up to about 150 kb and transferring them into plants. This vector is based on a BAC vector capable of carrying large DNA fragments and further contains elements for plant transformation such as T-DNA border sequences and a selectable marker as well as an origin of replication for Agrobacterium. The TAC vector (transformation-competent bacterial artificial chromosome) pYLTAC7 (Liu et al., 1999, Proc. Natl. Acad. Sci. USA 96: 6535-6540.) was also developed, which is capable of cloning DNA of up to about 80 kb and transferring it into plants. This vector is based on a high-capacity PAC vector (P1-derived artificial chromosome) using the replication mechanism of P1 phage and contains elements for plant transformation such as T-DNA border sequences and a selectable marker as well as an origin of replication for Agrobacterium. These vectors contain an origin of replication of (ori) from a plasmid existing as a single copy per cell in E. coli and Agrobacterium for the purpose of stably maintaining a large foreign gene. That is, they use an F factor on (BIBAC) or a P1 phage ori (TAC) as ori for E. coli and an Ri on from Agrobacterium rhizogenes (both BIBAC and TAC) as on for Agrobacterium. However, the use of an origin of replication from a single-copy plasmid is not necessarily essential, and vectors having an origin of replication (oriV) of an IncP plasmid known to exist as a few copies per cell such as pSLJ1711 and pCLD04541 were reported to be capable of stably maintaining plant genomic DNA fragments of more than 100 kb in size (Tao and Zhang (1998) Nucleic Acids Res 26: 4901-4909). In addition, pBIGRZ was also reported, which contains Ri ori in the versatile binary plasmid vector pBI121 (JPA Hei-10-155485).
Such vectors can be used to clone large DNA fragments far exceeding 50 kb, but involve complicated cloning operations. Cloning of large DNA requires skilled techniques and a considerable amount of time and labor. Transformation with BIBAC requires special Agrobacterium cells overexpressing virG or the like and results in a much lower transformation efficiency (the number of selected calli/inoculated leaf section) for fragments of 150 kb as compared with those of normal small vectors (Hamiltin et al., 1996, Proc Natl Acad Sci USA 93: 9975-9979, Shibata and Liu, 2000, Trend Plant Sci 5: 354-357). Thus, transformation of large fragments into plants with BIBAC or TAC is limited to a few specific examples of large fragments (e.g., Hamiltin et al., 1996, Proc Natl Acad Sci USA 93: 9975-9979, Liu et al., 1999, Proc Natl Acad Sci USA 96: 6535-6540, Lin et al., 2003, Proc Natl Acad Sci USA 100: 5962-5967, Nakano et al., 2005, Mol Gen Genomics 273: 123-129).
As described above, pCLD04541 is a cosmid of 29 kb in size, and therefore, the size of DNA fragments that can be cloned using a lambda phage packaging reaction is 10-20 kb. If cloning of larger DNA fragments is intended, a packaging reaction cannot be used as described above, and thus complicated cloning operations and a considerable amount of time and labor are required.
Recently, genetic markers based on DNA sequence polymorphisms or so-called DNA markers are used more and more frequently with the advance in genome studies of higher plants. Many attempts have been made to clone unknown genes of higher plants known only by their phenotypes on the basis of genetic map information using DNA markers, i.e., so-called map-based cloning. Generally, the basic protocol of map-based cloning is as follows.
1. Examine a relatively small segregating population with a set of DNA markers widely used for rough mapping of a candidate region on a chromosome.
2. Screen a large segregating population with a set of DNA markers newly designed for the particular region of the genome to narrow down the candidate region.
3. Determine the nucleotide sequence of the genetic region and guess a candidate gene.
4. Transfer a DNA fragment containing the candidate gene into a plant and determine the effect/function of the gene on the basis of the phenotype.
Many previous successful cases often involve narrowing down the genetic region to about 1-3 genes in step 3 and transferring several DNA fragments of several kilobases or less in step 4. However, it is not always easy to narrow down the genetic region. For example, it is often impossible to narrow down the genetic region to 150 kb or less in chromosomal regions near centromeres because of the low frequency of genetic recombination upon cross-hybridization. Even cases where narrowing down is possible often require repeating the operation of step 2 and therefore enormous amounts of time. Even if narrowing down to about 50 kb were possible, it would be very difficult to guess a candidate gene without strong information linking the phenotype to the gene sequence in step 3.
Thus, map-based cloning is relatively easy until the step of defining a candidate region including one to a few DNA fragments cloned by a BAC vector by narrowing down to some extent (to 50 kb to several hundreds of kilobases), but it is often technically difficult to further pursue the analysis to practically identify a gene, and even if it is possible, enormous amounts of labor and time are often required.